CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells

KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound 31 and V3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2's association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection. Author Summary KSHV infection of endothelial cells in humans leads into the development of Kaposi's sarcoma (KS). Hence, understanding of in vitro KSHV entry in endothelial cells is critical to develop strategies to control KSHV infection and KS. The de novo KSHV infection of endothelial HMVEC-d cells is initiated by its attachment to cell surface integrins, activation of cellular signals, and interaction with the receptor tyrosine kinase EphrinA2. This results in plasma membrane protrusions (blebs) in the lipid raft regions that engulf and internalize the virus, a process known as macropinocytosis. However, the identity of the molecule(s) coordinating the macropinocytic KSHV entry is not completely known. The present study identifies calcium and integrin-binding protein-1 (CIB1) as a key effector molecule promoting EphA2 associated signal events. CIB1 depletion by shRNA significantly reduced KSHV-induced bleb formation, activation of EphA2, Src, and Erk1/2, virus entry by macropinocytosis, productive trafficking, and infection. Our results also demonstrated that CIB1 plays a role in scaffolding EphA2 with cytoskeletal myosin IIA and alpha-actinin 4 during KSHV entry. Together, these studies reveal for the first time the role of CIB1 as a potential adaptor molecule in virus macropinocytic entry and indicate CIB1 as an attractive target to block KSHV entry and infection.